Light emitted and scattered from samples is widely used to determine their cellular or biological content. It is desirable to have a multi-colour detection system that allows analysis of a wide optical detection range of biological samples, with high resolution, instantaneously for a variety of assays in immunology, biochemistry, and haematology for example. Pre-defined algorithms combining multi-sensor outputs in a multi-colour detection platform would provide the end-user with the capability of running these assays without the need for detailed knowledge of the technology and with minimal interaction and analysing it with high resolution over an extended dynamic range.
Photodetectors are required to detect and quantify this light from a sample under test, when stimulated by some form of luminescence. Photodiodes, arrays of photodiodes, CCDs and other solid state sensors can be used to quantify this light; however Photomultiplier Tubes (PMT's) are the incumbent photodetector of choice to detect such light components. This is due to their low-end sensitivity and ability to resolve over the PMT's dynamic range due to their internal gain structure (>10 e6).
However, a number of problems exist in that PMT's are expensive, require very high voltage operation, are not suited to point of care diagnostic instruments and settings due to their size, require complex and expensive optical arrangements, with PMT based instruments requiring specialised and expensive maintenance. A PMT's practical optical dynamic range is typically 3 decades, so to adjust its optical range of sensitivity to quantify the light emitted or scattered from a sample requires the bias voltage to be adjusted by a skilled operator trained in the use of each specific instrument.
Due to recent advances in high gain semiconductor optical sensors such as avalanche photodiodes (APDs), Geiger-mode avalanche photodiodes and arrays of these Geiger-mode detectors (referred to as Silicon Photomultipliers or SiPMs for short) they are beginning to replace PMT's in such analysers. For example EP2293032A1, assigned to the applicant of the present invention and incorporated herein by reference, describes an integrated cytometric sensor system using SiPMs and other advances to solve such aforementioned issues. The main advantages relate to lower cost and size, lower voltage operation, faster start-up times, scope for increased semiconductor integration and lower maintenance requirements. These advances open the possibility of bringing advanced central laboratory techniques, such as the gold standard method of clinical flow cytometry, into decentralised point-of-care environments for screening of patients' blood for various infectious diseases, chronic and acute conditions, viruses and blood ailments.
Similar to PMTs, solid state sensors that contain an internal gain structure, like APDs and SiPMs, allow their region of sensitivity to be selected for the sample under test, by altering their operating voltage. This is controlled somewhat by setting their voltage bias with respect to their breakdown voltage.
By setting the bias voltage higher than the breakdown voltage (placing the SiPM in Geiger mode) these high gain semiconductor optical sensors can detect dimmer light, but saturate easily in the presence of bright light. By lowering the bias voltage towards the breakdown voltage or below it (placing the SiPM in its linear mode of operation), brighter light can be detected before saturation occurs, but the solid state sensor will be less sensitive to dimmer light as a consequence.
Lowering the bias of a high gain semiconductor optical sensor extends its dynamic range, enabling the detection of brighter light at the expense of reducing resolution. This is due to the reduction in the sensor's internal gain with reducing bias voltage and hence reduction of the optical sensor's output photocurrent and responsivity. This compromise between resolution and dynamic range limits the ability of an optical system using two or more sensors biased at different bias levels to resolve between biological samples or cells with similar but distinct high light level intensities, thereby limiting the range and/or resolution of the system. EP 2293032A1 uses such a method of adjusting the bias voltage of the sensor for wide dynamic range operation in an integrated multi-colour cytometric sensor based on high gain semiconductor optical sensors such as SiPMs. A problem with this method is that the reduction in bias will minimize the resolution of the system.
Additional systems where a multi-sensor approach is used to extend the dynamic range of an optical detection system is disclosed in US 2005/0151964 which outlines the expansion of its dynamic range by splitting the fluorescent light over multiple paths with different intensities onto multiple sensors/channels. It is then determined which channel is operating in its linear range and the output signal adjusted according to the intensity of light, using post processing techniques.
U.S. Pat. No. 5,491,548 similarly produces a wide dynamic range output from two optical sensors where a percentage of light is split onto the first sensor and the remainder is transferred onto the second sensor and the outputs are combined digitally to produce a composite signal. However this invention uses two different types of sensor to detect the light. Additionally, the use of a switch ensures that only the data from one of these sensors can be used at a time.
U.S. Pat. No. 6,355,921B1 describes a method where the output signals from multiple PMTs are combined to increase the dynamic as in the other prior art. Also the dynamic range of each PMT individually can also be increased by using a control circuit to combine the output from a low light level detection circuit and a similar bright light level detection circuit where needed. EP 1928167A1 involves using multiple detectors again and adjusting their parameters using a signal processing unit in a targeted manner. One detector is adjusted to a dynamic range for a maximum level of electromagnetic radiation expected, while the other detector is adjusted to a reduced dynamic range for small and middle signal levels in order to obtain a higher signal to noise ratio (SNR) for these. These systems rely on a number of physically different detectors with different surface area and active areas and cannot work using a single type of detector. Additionally the sensors are mounted as a single array and does not use a beam splitter.
It is an object of the invention to provide a system and method to overcome at least one of the above mentioned problems.